A major goal of modern medicine is to understand the mechanisms by which organisms develop and cells become muscle cells, liver cells, or even cancerous cells. Like many proteins participating in this complex process, even-skipped controls the development of the segmentation pattern in Drosophila by binding to regions of DNA near certain genes, and regulating the level of transcription from them. The long term objectives of our research are to understand, in a structural sense, how even-skipped recognizes specific DNA sites, and how it exerts an activating or repressing influence on RNA polymerase and its associated factors. Like many transcription factors, even-skipped contains a region consistent with DNA binding, the homeodomain, and regions consistent with repression/activation functions. The even-skipped homeodomain displays an unusual capacity to bind with similar affinities to both an A:T rich DNA site and a G:C rich DNA site. In order to achieve our objectives, the specific aims of this proposal are: 1) To determine the structures of even skipped homeodomain with A:T and G:C rich DNA sites by X-ray crystallography. X-ray data extending to 2.5A has already been collected from the A:T rich cocrystals. Attempts are underway to improve the 3.8A resolution of the G:C rich cocrystals by screening DNA fragments of different lengths and constructions. 2) To determine the structure of a fragment of even skipped, the "Pap" fragment, containing residues outside of the homeodomain. We will cocrystallize this fragment with DNA and determine its three dimensional structure, in order to better understand the role of residues outside of the homeodomain to DNA binding. 3) We will cocrystallize various mutants of even-skipped homeodomain with various DNA fragments. Our aim is to understand and design mutant proteins with altered DNA specificities. The mutants will be generated by site directed mutagenesis using PCR methods. 4) To determine the structure of the intact even-skipped protein. Our aims are to see how the repression/activation domains of even-skipped are folded up in three dimensions, and to determine how they may interact with the basal transcriptional machinery of the cells - to switch genes on and off. We will purify the protein in large quantities and crystallize it for structural studies.